Selectin Mediated Cell Adhesion
The migration of white blood cells from the blood to regions of pathogenic exposure in the body is called the inflammatory cascade. Cell adhesion events allow for specific binding of a leukocyte to the endothelium of the vessel that is adjacent to the inflammatory insult; such adhesion events counteract the high vascular shear forces and high blood flow rates that tend to keep the leukocyte circulating, and help guide the leukocyte to the required site.
Four families of vascular adhesion molecules are involved in the migration of leukocytes during the inflammatory response: (1) the integrin family, (2) the counterreceptors of the integrin family, the immunoglobulin superfamily, (3) the selectin family, and (4) the counterreceptors of the selectin family, specialized carbohydrates displayed by the sialomucin adhesion family.
Selectins are also known as "lectin cell adhesion molecules" (LEC-CAMs). Selectins are classified into three groups: L-selectin (LECAM-1, LAM-1, gp.sup.90.sub..sup.MEL , Leu-8, TQ-1, CD62L and DREG) is expressed on various leukocytes, and is constitutively expressed on lymphocytes, monocytes, neutrophils, and eosinophils. E-selectin (LECAM-2, CD62E and ELAM-1) is expressed on endothelium activated by inflammatory mediators. P-selectin (GMP-140, PADGEM, LECAM-3 and CD62P) is stored in alpha granules of platelets and Weibel-Palade bodies of endothelial cells and is also expressed on endothelium activated by inflammatory stimuli. All members of the selectin family appear to mediate cell adhesion through the recognition of carbohydrates.
The current concept of leukocyte extravasation is based on the consecutive action of several adhesion molecules located on the surface of leukocytes and the endothelium. Lymphocyte extravasation is initiated by the interaction of members of the selectin family and their oligosaccharide-containing counterreceptors. For a review of the current knowledge on lymphocyte adhesion, see e.g., Springer, T. A., Annu. Rev. Physiol. 57:827-872 (1995).
All selectins bind to sialyl Lewis x (NeuNAc.alpha.2-3Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc) (sLe.sup.x or sLex) and sialyl Lewis a (NeuNAc.alpha.2-3Gal.beta.1-3(Fuc.alpha.1-4)GlcNAc) (sLe.sup.a or sLea) as well as related carbohydrate sequences (Bertozzi, C., Chemistry and Biology, 2:703-708 (1995)). L-selectin-dependent recognition precedes normal lymphocyte extravasation into peripheral lymph nodes (Gallatin, W. M. et al., Nature 303:30-34 (1983)) and into sites of inflammation (Ley, K. et al., Blood 77:2553-2555 (1991)), both of which are impaired in L-selectin deficient mice (Arbones, M. L. et al., Immunity 1:247-260 (1994)).
Several glycoproteins have been shown to act as counterreceptors for L-selectin. A common denominator for the cloned ligands GlyCAM-1, CD34, MAdCAM-1 and PSGL-1 (Baumhueter, S. et al., Science, 262:436-438 (1993); Briskin, M. J. et al., Nature, 363:461-464 (1993); Lasky, L. A. et al., Cell 69:927-938 (1992); Walcheck, B. et al., J. Clinical Investigation 98:1081-1087 (1996)), is the mucin type protein core rich of 0-linked glycan decorations which are crucial for the selectin recognition. The glycosylation of GlyCAM-1 and PSGL-1 has been characterized in greater detail, among other saccharides these proteins have been shown to carry sulfated sLex and sLexLexLex epitopes, respectively (Hemmerich, S. et al., J. Biol. Chem. 270:12035-12047 (1995); Wilkins, P. P. et al., J. Biol. Chem. 271:18732-18742 (1996)).
High endothelial cells in peripheral lymph nodes express sialyl Lewis a and sialyl Lewis x (sLea and sLex) epitopes (Paavonen and Renkonen, Am. J. Pathol. 141:1259-1264(1992); Munro, J. M. et al., Am. J. Pathol. 141:1397-1408 (1992); Sawada, M. et al., Biochem. Biophys. Res. Comm. 193:337-347 (1993)) which are parts ofthe L-selectin counterreceptor. The endothelial cells in several other locations are sLea and sLex negative, but inflammatory stimuli can induce previously negative endothelium to express these oligosaccharide structures de novo (Turunen, J. et al., Eur. J. Immunol. 24:1130-1136 (1994)). It has been shown that cultured endothelial cells possess the machinery to generate at least sLex, since they have several functional .alpha.2,3 sialyl- and .alpha.1,3 fucosyltransferases, enzymes involved in generating sLex from (poly)lactosamines (Majuri, M. et al., Eur. J. Immunol. 24:3205-3210 (1994)).
A number of studies have proposed that selectins are involved in a wide variety of acute and chronic inflammatory conditions in many tissues.
Being essential to the early phases of leukocyte extravasation cascade, the interactions of selectins and their sLex-containing counterreceptors offer an attractive site for anti-inflammatory interventions (Ley, K. and Tedder, T., J. Immunol. 155:525-528 (1995); Springer, T. A., Annu Rev. Physiol 57:827-872 (1995); Tedder, T., et al, FASEB Journal 9:866-73 (1995)). Monovalent sLex glycans have been shown to inhibit L-selectin mediated lymphocyte binding in vitro (Turunen, J. et al., Eur. J. Immunol. 24:1130-1136 (1994)), and they also inhibit PMN extravasation in animal models of acute inflammation and reperfusion injury (Lefer, D. J. et al., Circulation 90:2390-401 (1994)).
Polylactosamines carrying single epitopes of sLexLex- and sLexLexLex-type appear to be recognized by E- and P-selectins with higher affinity than analogous oligosaccharides bearing single sLex-units (Patel, T. P. et al., Biochemistry 33:14815-24 (1994); Stroud, M. R. et al., Biochemistry 35:770-778 (1996); Wilkins, P. P. et al., J. Biol. Chem. 271:18732-18742 (1996)).
U.S. Pat. No. 5,352,670 to Venot et al. discloses a method for the enzymatic synthesis of an .alpha.-sialylated oligosaccharide glycoside using sialyltransferase, a CMP-sialic acid analogue as the sialic acid donor and an oligosaccharide glycoside acceptor molecule, having a .beta.Gal(1-3).beta.GlcNAc or .beta.Gal(1-4).beta.GlcNAc disaccharide on the nonreducing terminus.
International Patent Publication No. WO 95/03059 (Gaeta et al.) discloses a synthetic saccharide that contains two glycosidically linked sLex moieties, that are useful in blocking cellular adhesion, especially by inhibiting E-selectin binding. These sLex containing oligosaccharides are synthesized on a galactose backbone.
International Patent Publication No. WO 97/12892 (Renkonen, O. and Renkonen, R.) discloses synthetic multivalent sLex containing polylactosamines and their use to block lymphocyte binding to correspondent oligosaccharides on the endothelial surface.